DESCRIPTION (Applicant's Abstract): Pulmonary surfactant is a lipid and protein complex present in the alveolar compartment that resides at the interface between the human lung and the external environment. The constituents of pulmonary surfactant are the first biological components of the host that aerosolized mycobacteria contact upon reaching the alveolar compartment. Surfactant protein A (SP-A) and surfactant protein D (SP-D) are two abundant proteins present in the extracellular fluid of the alveolus that directly interact with mycobacteria and the target cell for replication, the alveolar macrophage. The interactions of mycobacteria with SP-A and SP-D comprise the very earliest events in the process of invasion and infection by the organism. The goals of this project are to understand the physical interactions between SP-A and SP-D and mycobacteria at these very early stages, and determine how this influences the process of bacterial entry and replication within the macrophage. In order to address these goals experimentally, we will use structural mutants of SP-A and SP-D to examine how specific domains of the proteins interact directly with the mycobacteria. The repertoire of mutants we have include point mutations within each of the four major structural domains, and chimeric proteins between SP-A and SP-D and Mannose Binding Protein A. These studies will enable us to determine which protein domains are required for mycobacterial recognition. We will also determine the role of SP-A and SP-D in promoting the successful entry of mycobacteria into the macrophage for replication. The SP-A and SP-D structural variants will be used to map which protein domains are required for the interactions with the macrophage that promote mycobacterial infection. In order to understand the interactions of the mycobacteria with SP-D in more detail, we will prepare derivatives of a major mycobacterial membrane envelope component, mannosyl-lipoarabinomannan, and cocrystallize them with a truncated version of the human SP-D molecule. These latter studies will enable us to define at the atomic level how the mycobacterial envelope interacts with the surfactant proteins. Collectively, the proposed studies will provide us with a detailed map of interactive sites between the surfactant proteins and mycobacteria and macrophages that will create new opportunities for developing agents that interfere with the infection process.